SUMMARY Opiate abuse is a significant risk factor for HIV-1 infection and several studies have shown that, in combination, opiates and HIV-1 lead to significantly greater damage to the brain. Thus, a new combinatory strategy is needed to impede HIV-1 infection and mitigate opiate effects on the CNS. In spite of significant advances in anti-retroviral therapy (ART), the elimination of HIV-1 CNS reservoirs remains a formidable task. This is mainly attributed to the integration of the HIV-1 proviral DNA into the host genome causing viral latency in the reservoirs, including the brain. Further, the inability of ART to penetrate the BBB after systemic administration makes the brain one of the dominant HIV reservoirs. Thus, elimination of HIV-1 from the brain remains a clinically daunting and key task in the cure of HIV-1/opioid CNS disease. Most recently, we (Dr. Khalili's lab at Temple University) developed an RNA directed gene-editing strategy using Cas9/gRNA that successfully eliminates entire integrated copies of the HIV-1 genome from the host chromosome. However, delivery of this powerful Cas9/gRNA complex across the BBB is limited and an effective method for delivery and release of Cas9/gRNA is critically required to eliminate the HIV reservoir in the brain. Our laboratory (Dr. Nair's team at Florida International University) has recently patented (US patent: US20130317279 A1 and WO patent: PCT/US2013/068698) technology involving novel magneto-electro nanoparticle (MENP) based drug delivery system, which offers capability of on-demand drug release across the BBB. The collaboration of these two laboratories provided preliminary evidence that Cas9/gRNA binds to MENP, navigated across the BBB by magnetic force, and on-demand release of functionally active Cas9/gRNA by external AC stimulation. We provide evidence that morphine induced activation of HIV infection could be mitigated by methylnaltrexone (MTNX) ( receptor antagonist). In this multi-PI application we hypothesize that efficient nanoformulations (NFs) containing Cas9/gRNA and MTNX can serve as an effective carrier to deliver Cas9/gRNA targeting HIV-1 across the BBB for the recognition and complete eradication of the HIV reservoir in brain and to treat/prevent neurological deficits observed in morphine-using HIV infected subjects. To test our hypothesis, we propose to refine our design method, and develop, characterize, and evaluate the delivery and on-demand release of Cas9/gRNA using an in vitro BBB-HIV infection model (Aim #1). Next, we will evaluate and pre-screen the in vivo efficacy of the developed NFs in excising integrated copies of HIV DNA in Tg26 transgenic mice harboring the entire viral genome (Aim #2). In Aim #3 we will develop and use BLT mouse model to validate and assess the in vivo efficacy of the MENP-Cas9/gRNA NFs to recognize and eradicate latently infected HIV-1 reservoirs. Finally, in Aim #4 we will examine the in vivo efficacy of the most pre-screened NFs in a BLT morphine mouse model to assess the potential excision of HIV-1 proviral DNA and morphine induced reactivation of latent HIV infection and to reverse neurological deficits by NFs containing MNTX.